Conformational dynamic af the adenylation domain in tyrocidine synthetase 1 followed by fluorescence spectroscopy

Šprung, Matilda (2014) Conformational dynamic af the adenylation domain in tyrocidine synthetase 1 followed by fluorescence spectroscopy. Doctoral thesis, Faculty of Science > Department of Chemistry.

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Abstract

Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes that catalyze the synthesis of biologically active peptides in an array of transpeptidation reactions. The process of biosynthesis starts with the adenylation (A) domain, which in the first half reaction catalyzes the synthesis of the aminoacil-adenylate, and in the second amino acid transfer to the adjacent tiolation (T) domain. Crystal structures of various A-domains show large structural rearrangements accompanying the catalytic process that are in correlation with the coordination of catalytic steps and interaction with the adjacent T-domain. Here we report the investigation of conformational changes in the A-domain of tyrocidine synthetase 1 (TycA-A) probed by fluorescence spectroscopy. TycA-A comprises five tryptophan residues in the primary sequence: W227, W301, W323, W376 and W406. Trp side chain accessibility was probed using TycA-A homology models in two distinctive protein conformations. Fluorescent quenching with acrylamide, carried out on the wild type and mutant TycA-A proteins caring individual Trp substitutions, revealed two independent groups of fluorophores. The Results show that the presence of reaction substrates has affected only the W227 residue which can serve as an intrinsic fluorescent probe. The effect of noncognate amino acid substrates on the TycA-A conformation was probed on mutant TycA-A bearing only one fluorescent Trp residue. The Results show a suboptimal protein conformation and reduced protection of the reaction intermediate from hydrolysis. Conformational changes induced by substrate binding were probed by limited proteolysis and differential scanning fluorimetry methods.

Item Type: Thesis (Doctoral thesis)
Keywords: adenylation domain, nonribosomal peptide synthetases, conformational dynamics, tyrocidine synthetase 1
Supervisor: Pavela-Vrančić, Maja
Date: 2014
Number of Pages: 117
Subjects: NATURAL SCIENCES > Chemistry
Divisions: Faculty of Science > Department of Chemistry
Depositing User: mag. bibl. Ana Šafran
Date Deposited: 18 Dec 2014 16:18
Last Modified: 15 Jun 2015 12:57
URI: http://digre.pmf.unizg.hr/id/eprint/3557

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